Plant genomes contain substantial amounts of 5-methylcytosine. Up to 20-30% of the cytosines are methylated in the nuclear genome of many flowering plants. As in other organisms, methylation of cytosine residues in plants occurs post-replicatively through the action of cytosine-DNA methyltransferases. Plant DNA methyltransferases have been characterized biochemically, and plant genes encoding these enzymes have been isolated by virtue of their similarity to their mammalian counterparts.
Investigations of native plant genes and transgenic plants containing foreign genes have found a general correlation between transcriptional inactivity and increased DNA methylation, consistent with evidence from mammalian systems. This evidence supports a role for cytosine methylation in maintaining transcriptional states.
The plant's need for developmental plasticity and environmental interaction suggests that plants extensively employ epigenetic regulatory strategies. Such strategies rely on heritable, often reversible, changes in access to the underlying genetic information, but not alteration of the primary nucleotide sequence. As one example, the alteration of DNA methylation is expected to perturb plant development significantly, provided that differential DNA methylation is an important component of epigenetic regulation in plants.
One paradigm linking DNA methylation and developmental regulation comes from work on the mouse, where average genome cytosine methylation levels in embryonic lineages drop sharply in the early cleavages following fertilization, then rise again around the time of implantation. In plants, a similar pattern has been observed in studies of DNA methylation content in pollen and post-embryonic tissue of varying age. Information from such studies indicates that there is a gradual rise in 5-methylcytosine levels in post-embryonic tissues produced by meristems at positions further from the base of the plant (i.e., tissues of increasing age). Genetic studies of transposon systems in maize also demonstrate an age-dependent gradient of increasing epigenetic modification, which is correlated with DNA methylation.
Both biochemical and genetic approaches have been taken to alter DNA methylation in eucaryotic organisms. Methylation inhibitor treatments have induced developmental abnormalities in many plant species. Transgenic plants expressing antisense molecules specific for a native cytosine methyltransferase gene have been found to exhibit genomic hypomethylation, presumably due to the antisense interference with expression of the gene.
In another approach, mutants of Arabidopsis thaliana have been isolated, which show a decrease in DNA methylation (ddm) resulting in reduced nuclear 5-methylcytosine levels. The best characterized mutations define the DDM1 gene. Homozygotes carrying recessive ddm1 alleles contain 30% of the wild-type levels of 5-methylcytosine. The ddm1 mutations do not map to the two known cytosine-DNA methyltransferase genes of A. thaliana, nor do they affect DNA methyltransferase activity detectable in nuclear extracts (Kakutani et al., Nuc. Acids Res. 23: 130-137, 1995). In addition, ddm1 mutations do not appear to affect the metabolism of the active methyl group donor, S-adenosylmethionine (Kakutani et al., 1995, supra).
For the foregoing reasons, the DDM1 gene product is likely to be a novel component of the DNA methylation system, or involved in determining the cellular context (e.g., chromatin structure, subnuclear localization) of the methylation reaction. Consequently, it would be a clear advance in the art of plant molecular and cellular biology to identify and isolate the DDM1 gene and/or its encoded protein. Such a gene and protein would find utility for the purpose of modifying the methylation status of a selected genome and thereby altering one or more regulatory features of gene expression from that genome.